A yeast thioredoxin, dependent peroxidase (TPx1) was previously identified. A second thioredoxin, dependent peroxidase, named TPx2, has now been purified from yeast, and the corresponding cDNA has been cloned and sequenced. Comparison of the predicted sequence of 176 amino acids of TPx2 with that of the 195 residues of TPx1 revealed no substantial homology with the exception of a short segment preceding Cys 62 of TPx2. TPx1 preferentially reduces H2O2 rather than alkyl hydroperoxides, whereas TPx2 shows the reverse specificity. TPx2 contains three cysteine residues at positions 31, 62, and 120. Cys 62- SH constitutes the site of oxidation by peroxides and the oxidized Cys 62 reacts with the Cys 120- SH group of another TPx2 molecule to form an intermolecular disulfide. The TPx2 disulfide can then be reduced by thioredoxin, but not by glutathione. Thus, TPx2 mutants lacking Cys 62 or Cys 120 showed no detectable TPx activity, whereas mutation of Cys 31 had no effect. An antioxidant function of TPx2 in intact cells was demonstrated. Although most Prx family members contain two conserved cysteines, seven Prx proteins from various organisms contain only one conserved cysteine residue. Thus, members of the Prx family can be divided into two subgroups, 1- Cys and 2- Cys. Human 1- Cys Prx has now been shown to reduce H2O2 with electrons provided by dithiothreitol. Furthermore, human 1- Cys Prx transiently expressed in NIH 3T3 cells was able to remove intracellular H2O2 generated in response to PDGF. The conserved Cys 47-SH group was shown to be the site of oxidation by H2O2. Thus, mutation of Cys47 to serine abolished peroxidase activity. Moreover, the oxidized intermediate appears to be Cys-SOH. In contrast to TPx, the Cys-SOH of 1, Cys Prx does not form a disulfide. Neither thioredoxin, which reduces the disulfide of TPx, nor glutathione, which reduces the Cys-SeOH of oxidized glutathione peroxidase, was able to reduce the Cys-SOH of 1- Cys Prx and consequently could not support peroxidase activity.